Deregulation of the control of cell cycle is an important event in malignant transformation of tumoral cells and is a common characteristic to all types of cancer, where commonly is observed an alteration in genes controlling the cell cycle as well as proliferation.
In cell cycle control, there are 2 checkpoints which are critical: transition between phases G1-S and transition between phases G2-M. The principal components of the machinery of the cell cycle are cyclins and its correspondent ciclyn dependent kinases (CDK). These complexes CDK/cyclin are activated and inactivated sequentially, thus regulating the different phases of the cell cycle.
CDK/cyclins are subject to various mechanisms of control, including association with inhibitory proteins, such as p15lnk4b/p16lnk4a or p21Waf1/p27Kip1, activating phosphorylation on Thr160/161, activating CDK and inhibitory phosphorylation on Th14 and Tyr15 by Wee1 and Myt1 kinases.
Another control mechanism is exerted by CDC25 phosphatases, which are key in the control of the cell cycle in eukaryote cells, in normal conditions as well as in response to cell damage, and whose over expression is associated to a wide variety of cancer.
The physiological substrate of CDC25 phosphatases are CDKs. The complex CDK/cyclin is maintained inactive by phosphorylation of the minor lobule of CDK subunit, and in normal conditions, during cell division, a member of this class of phosphatases dephosphorylates CDK, thus allowing progression of the cell cycle.
Three isoforms of CDC25 phosphatases have been identified in mammals: Cdc25A, Cdc25B, and Cdc25C.
Human Cdc25 proteins have between 423 and 566 aminoacids, with a conserved catalytic domain compared to the regulatory regions, which are more diverse and subject to splicing events. These variability generates in humans 3 variants for Cdc25A, 5 for Cdc25B, and 5 for Cdc25C. Splicing variants are particularly relevant for Cdc25B, since its most active isoform, Cdc25B2, is the only one detected in primary fibroblasts, while at least 3 different variants of splicing are present in immortalized fibroblasts.
The activity of these phosphatases is highly regulated during normal division of cells and in response to the activation of checkpoints, in order to assure a correct functioning of CDC/cyclin activity.
Considering all the mentioned issues, is not surprising to find Cdc25B reported in various types of cancer, suggesting its participation in pathogenesis and progression of malignant transformation of cells. It is known that Cdc25B is over expressed in 72% of pancreatic cancer cases, and together with gastric cancer, are the only 2 types of cancer which only over express Cdc25B exclusively, and no other isoform of this phosphatase.
In particular, pancreatic cancer is the fifth cause of death in many western countries, being one of the most aggressive and with the worst prognosis: a survival rate for 5 years of only 5%. Currently, the only treatment is surgery, but only 10% of the patients suffering from such a form of cancer are suitable candidates, while the remaining 90% has only an average of 4 to 5 months of survival time.
Considering only the 10% of patients which can receive a surgical treatment, less than 10% shows a survival rate higher than 5 years.
Currently, gemcitabine is the chemotherapeutic agent of first line to treat pancreatic cancer. Nevertheless, even when applying this agent, the survival is only 1 year in average. Thus, new strategies and ways of treatment need to be developed, being gene-therapy one interesting and promising option.
Up to date, nearly 66% of clinical trials of gene-therapy are directed to treat cancer. Among the key issues addressed by these therapies, are: increasing antitumor activity of immune cells by using genes coding cytokines, increasing the immunogenicity of the tumor by application of tumor antigens, using a “suicidal” gene which grants increased sensibility to certain pharmaceutical compounds. Other strategies include the blocking of oncogenes by using antisense genes, using tumor suppressor genes (p53), transfection of genes with antiangiogenic effect, using genes to increase resistance to certain pharmaceutical compounds to decrease toxicity of chemotherapy, and use of oncolytic viruses.
Among the oncolytic viruses, the two most used systems are retroviruses and adenoviruses, and adenoviruses are the system of choice given a safety issue, since adenoviruses are not oncogenic.
There are 51 human serotypes of adenoviruses, which are divided in 6 species, A to F. These are double strand linear DNA, with 35-36 kb length, contained in a protein capsid, which is formed by 240 hexagonal capsomers (hexons) covering the faces and edges, comprising the most abundant component, and 12 pentagonal (pentones) located in the vertices, from which extensions or fibers emerge.
Pentons and fibers are responsible of the interaction cell-virion. The fiber comprise a globular domain (knob) which serves as the main binding site “Coxsakie and adenovirus receptor” CAR, while the penton comprises a RGD domain (Arg-Gly-Asp), which interacts with the cell surface integrins, thus producing the endocytosis of the virion via clatrin vesicles.
The first generation of adenoviral vectors was able to infect tumoral cells, but unable to replicate, since E1A gene, responsible for replication, was deleted from its genome. Unfortunately, non-replicant viruses were found to be ineffective in achieving a proper therapeutic response. Thus, a new generation of conditionally replicative adenoviruses (CRAds) was created.
Two types of CRAds can be distinguished, one with a mutation or deletion of E1A or E1B gene, and the other where E1 genes are under transcriptional control of a tumor specific promoter (TSP). The replicated viruses can infect neighboring cells causing lysis, which will continue as long as the tumor specific promoter is active. Thus, CRAds are not only a carrier for gene transfer, but also an active therapeutic agent.
Currently, in China, there is an oncolytic adenovirus, (H101, Shangai Sunway Biotech), which combined with fluorouracil (5FU) and cisplatin has been accepted as a standard treatment for refractory nasopharyngeal cancer.
In terms of pancreatic cancer, this is the one with the highest number of mutations, compared to all other tumors. Genetic and epigenetic alterations have been reported, including mutations in protooncogene K-Ras and suppressor genes such as p53, Smad4/DPC4 and p16/CDKN2A. These alterations result in an uncontrolled progression of the cell cycle, with high resistance to cell arrest and apoptosis. Furthermore, there is an over expression of growth factors and its receptors in a significant amount of these tumors.
It is also known that expression levels of Cdc25B are increased in pancreatic cancer, but not in chronic pancreatitis or normal pancreas. In contrast, Cdc25A and Cdc25C do not show difference among normal samples and tumoral samples. Furthermore, in normal tissue, Cdc25B is expressed in 8% of cells, while in tumoral tissue the level of expression is 48%. Of the tumoral cells, Cdc25B is found in the nucleus as well as in cytoplasm.
Thus, Cdc25B is a good candidate to be used as promoter to direct the expression of adenoviral replication in tumoral cells.
A further requirement to have a proper adenovirus therapy for cancer is having efficient gene transfer. Thus, an important issue is the high variability of the expression receptor CAR in tumoral cells.
Many studies show that pancreatic cancer cells have a low expression of CAR; therefore diverse approaches need to be considered to modify or reduce this deficiency, including genetic modifications altering the capsid or using redirecting complexes, and also reducing directing to hepatocytes, which present high expression of CAR.
One approach is to modify the structure of the fiber of the adenovirus, substituting the binding protein to the receptor CAR (knob/fiber) with one of a different serotype, thus producing chimera adenoviruses, significantly improving the adenovirus infectivity.
Modification of the HI loop in the knob region of the fiber, with RGD-4C motifs has shown an increased infectivity of cells expressing low levels of CAR, compared to a native Ad5 fiber. This improvement could be explained by the interaction of integrins, usually over expressed in tumoral cells, with the modified fiber. It has also been shown that replacing the knob domain in Ad5, whose primary receptor is CAR, with an Ad3 knob domain, whose primary receptor are CD46, CD80, and CD86, increases the infectivity of cells showing a low expression of CAR.
US patent U.S. Pat. No. 6,033,856 discloses the promoter of cdc25B gene, a process to find these promoters and method of use in pharmaceutical compositions. The document describes murine sequences, and mentions that the sequences can be human, but the human Cdc25B sequence is not disclosed.
WO2010097419 describes an adenovirus with conditional replication wherein E1A expression is controlled by the promoter of the urokinase plasminogen activator (uPAR) or a fragment thereof. It is also mentioned that this adenovirus can be used in the preparation of a medicine for the treatment of cancer, in particular pancreatic cancer. Nevertheless, this document does not mention nor suggest using the promoter of the Cdc25B gene to produce a new oncolytic adenovirus.
WO0067576 describes a serotype 5 adenovirus with an increased infectivity and conditional replication, wherein these characteristics are the product of a modification in the knob domain of the fiber. It is also mentioned that the early genes are conditionally controlled, thus offering replication limited to specific cell types. Nevertheless, this document does not mention nor suggest an oncolytic adenovirus comprising the Cdc25B promoter, or the modification of the knob domain as described in the present invention.
US2010233125 discloses a serotype 5 adenovirus, whose fiber has been replaced with the fiber of serotype 3adenovirus, and E1A and E1B are regulated by exogenous transcriptional regions. The adenovirus described is cytotoxic or oncolytic and can be used in the treatment of tumors. A process for its preparation and a medicine is also disclosed. Nevertheless, this document does not mention the use of a promoter sequence, such as Cdc25B, as disclosed in the present invention.
US20100297072 describes a pharmaceutical kit, comprising an oncolytic virus, and an immunostimulant drug. The document further describes a treatment method, but it does not describe an orthotopic cancer model, nor does it suggest the combination of an oncolytic adenovirus comprising a Cdc25B promoter combined with a chemotherapeutic agent.
US20010044420 describes the combination of gene therapy based on p53 gene and gemcitabine. Different cell lines are evaluated in terms of decrease in proliferation, but as will be seen in this application, the doses used for the viral vector are higher than the ones considered in the present invention, as well as the promoter considered to direct the replication of the virus.
US20090317456 describes a combination of gene therapy and an antiangiogenic agent. There is no description of an adenovirus similar to the description found in the present invention, wherein the oncolytic virus is specifically designed to target cancer combined with a chemotherapeutic agent.
US20070281041 describes a combination of MDA-7 which can be delivered by a virus, and a EGFR inhibitor, wherein the combination for cancer treatment can further comprise an anticancer drug, nevertheless, there is no mention nor suggestion for the treatment or use of the combination of an oncolytic adenovirus and a chemotherapeutic agent to treat a cancer characterized by Cdc25B over expression.
None of the documents found in literature report an oncolytic adenovirus, which is directed to cancer, characterized by an over expression of Cdc25B, and which combined with a chemotherapeutic agent, such as gemcitabine, produces a synergistic effect in the reduction of tumor size.